Fungemia is associated with high morbidity and mortality rates. Various techniques have been introduced to diagnose fungemia such as conventional broth culture medium, by blind or macroscopic subculture, vented and aerated biphasic culture medium, lysis filtration agitated culture medium, radiometric and nonradiometric methods, lysis centrifugation method, etc..[1] Of these, lysis-centrifugation technique seems to be a reasonable alternative, which combines the dual advantage of an increased yield with a short detection time as compared to the conventional blood culture system.[2] Lysis centrifugation is commercially available as isolator system, it can also be done as manual technique[3] for which limited studies are available. The present study is aimed to evaluate manual lysis centrifugation technique for detection of fungemia.

Ninety six patients were studied at a tertiary care general hospital in Mumbai between October 2004 and February 2006. Patients with risk factors such as long-term use of broad spectrum antibiotics, prolonged hospitalization, central and peripheral line insertion, respiratory ventilation, presence of urinary catheter, use of cytotoxic drugs, prematurity, and low birth weight were included in the study. Blood samples from each patient were collected and processed by the two techniques; conventional blood culture and manual lysis centrifugation (MLC). Conventional technique was done by inoculating blood into blood culture bottles containing brain heart infusion broth. The bottles were incubated at 37 0 C and were shaken periodically. On 3rd, 5th, and 7th day, subcultures were done on Sabouraud Dextrose Agar (SDA) slants.[1],[4] MLC technique was followed as described by Bianchi et al.[3] Blood samples were collected in autoclaved centrifuge tube containing isotonic saline solution with 5% saponin and 0.4% sodium salt of polyanethol sulphonic acid. The contents were mixed by shaking. Having kept at room temperature for 1 hour, the tubes were centrifuged at 3000 rpm for 30 minutes. The supernatant was removed and sediment was inoculated onto SDA and Brain Heart Infusion Blood Agar (BHIA) in duplication using aseptic precautions. One set of SDA and BHIA was incubated at room temperature and other set of the two culture media at 37 0 C for 6 weeks. Fungal growth was identified using standard mycological techniques.[5] All the fungal isolates were of Candida spp. Candidaemia was diagnosed by isolation of Candida spp. from at least two blood culture samples or single blood culture sample with supportive clinical features. Statistical analysis was done using Chi-square test on statistical software Minitab 14. To test the statistical significance of yield of Candida and time required for its growth with both the techniques, Chi-square test was done and exact P -values were obtained ( P ≤ 0.01 was considered significant).

Of the 96 patients studied, 32 (33.3%) had fungemia. C. albicans was isolated in 17 (53.1%), C. parapsilosis in six (18.8%), C. tropicalis in five (15.6%), and C. glabrata in four (12.5%) patients by either or both techniques. Of the 32 culture-positive specimens, 17 (53.1%) isolates had grown by both the techniques, 12 (37.5%) had grown by MLC, while three (9.4%) by conventional technique. Hence, 20 (62.5%) were positive by conventional technique whereas 29 (90.62%) were positive by MLC. Thus, manual lysis centrifugation was found to be superior technique than conventional technique in that the yield of Candida was more with the former technique ( P -value 0.008). The time required to detect fungal pathogen by both the techniques were also observed. Maximum number of isolates grew in first week by both techniques, a total of 18 (90%) grew in first week by conventional method and 28 (96.5%) by MLC technique. It was observed that only two (10%) isolates were recovered within 48 hours by conventional technique, while nine (31%) isolates were recovered by MLC technique within 48 hours [Table 1]. The time required for growth of Candida by MLC is less though this difference was not found to be statistically significant ( P -value 0.083). MLC technique appeared to be a superior technique to recover C. albicans from blood in this study. Eleven (64.7%) isolates of C. albicans grew by conventional technique and 17 (100%) isolates grew by MLC technique. The difference was found to be statistically significant ( P -value 0.01). The number of the nonalbicans species recovered was however less and more specimens need to be studied of nonalbicans candidaemia for authentication.

From our study, we are of the opinion that manual lysis centrifugation technique provides a more sensitive and rapid means of detecting organisms associated with fungemia. This finding is especially relevant for resource restricted setting where newer techniques are not available.